Search

The majority of variants of unknown clinical significance (VUCS) in the CFTR gene are missense variants. While change on the CFTR protein structure or function is often suspected, impact on splicing may be neglected. Such undetected splicing default of variants may complicate the interpretation of genetic analyses and the use of an appropriate pharmacotherapy.We selected 15 variants suspected to impact CFTR splicing after in silico predictions on 319 missense variants (214 VUCS), reported in the CFTR-France database. Six specialized laboratories assessed the impact of nucleotide substitutions on splicing (minigenes), mRNA expression levels (quantitative PCR), synthesis and maturation (western blot), cellular localization (immunofluorescence) and channel function (patch clamp) of the CFTR protein. We also studied maturation and function of the truncated protein, consecutive to in-frame aberrant splicing, on additional plasmid constructs.Six of the 15 variants had a major impact on CFTR splicing by in-frame (n = 3) or out-of-frame (n = 3) exon skipping. We reclassified variants into: splicing variants; variants causing a splicing defect and the impairment of CFTR folding and/or function related to the amino acid substitution; deleterious missense variants that impair CFTR folding and/or function; and variants with no consequence on the different processes tested.The 15 variants have been reclassified by our comprehensive approach of in vitro experiments that should be used to properly interpret very rare exonic variants of the CFTR gene. Targeted therapies may thus be adapted to the molecular defects regarding the results of laboratory experiments.

Because CFTR gene studies now represent one of the most frequent genetic analyses routinely performed worldwide, the number of rare CFTR variants identified in various clinical situations, regularly increases. To provide appropriate diagnosis and prognosis to CF patients as well as appropriate genetic counseling to families, the clinical impact and the phenotypic spectrum of variants identified by diagnostic techniques need to be characterized. Three complementary locus specific databases, called CFTR1, CFTR2 and CFTR-France were developed to address these issues. Besides, the growing knowledge of the CF pathophysiology and the technical evolution in molecular biology allowed to identify candidate modifier genes, regulatory loci, epigenetic profiles and trans-regulators that could help to refine genotype-phenotype correlations at the individual level. These different factors may contribute to the large phenotypic variability between patients with CF, even when they carry identical CFTR variants, regarding lung function, meconium ileus susceptibility or the risk for developing CFTR-related diabetes and liver disease. Finally, the availability of new therapies that target the CFTR protein for numbers of CF patients led to the identification of 'good' and 'poor' responders, thus raising questions of pharmacogenetics factors that may influence treatment efficiency as a novel feature of the complexity of CF patients' management. © 2020 French Society of Pediatrics. Published by Elsevier Masson SAS. All rights reserved.

International audience; Alumina production from bauxite ore gives rise to a large amount of liquid and solid waste since the production of 1 kg of alumina involves the same weight of solid residue, the well-known red muds enriched in iron and heavy metals. Up to now, without any valorization of by-products, the red muds have been stored in artificial ponds which are a potential source of pollutants for the surroundings due to the uplift of red dust under strong wind conditions. The goal of the current work is to perform an in situ study of the transfer of red mud micro particles in the atmosphere and in the terrestrial environment around the red mud disposition basins. To this end, the activity concentration of NORM (Naturally Occurring Radioactive Materials such as U-238, decay products and Th-232) determined in plant leaves (Quercus robur and leaf vegetables) and in grains (wheat) is compared with the activity of the atmospheric micro particles, both taken in the environment of the red mud disposition basins (100-1,500 m). The activity ratios of NORM in vegetation and atmospheric micro particles are also compared to the ratios of potential sources of dust, such as soil and, above all, the red mud and bauxite emitted from the basins and from the piles, respectively.The increase in Th-232 and U-238 activity concentration in a few tree leaves (by a factor of 9 and 4, respectively) and in some aerosol samples is accompanied by a decrease in U-238/Th-232 activity ratio for these matrices. Low U-238/Th-232 is also observed in bauxite and red mud. This suggests that the airborne particles emitted by bauxite piles and red mud basins first contaminate the atmosphere and then the leafs surfaces, after deposition. Locally produced foodstuffs at a distance greater than 1,000 m from the ponds do not show any excess radionuclides, suggesting the low influence of airborne particles from the alumina production. Further variations in the Po-210/Pb-210 ratio in the plants and the atmospheric micro particles are consistent with the variation over time of these radionuclides coming in all likelihood from the atmosphere rather than from the aluminum industry.

International audience; BACKGROUND:The CFTR genotype remains incomplete in 1% of Cystic Fibrosis (CF) cases, because only one or no disease-causing variants is detected after extended analysis. This fraction is probably higher in CFTR-Related Disorders (CFTR-RD). Deep-intronic CFTR variants are putative candidates to fill this gap. However, the recurrence, phenotypic spectrum and full molecular characterization of newly reported variants are unknown.METHODS:Minigenes and analysis of CFTR transcripts in nasal epithelial cells were used to determine the impact on CFTR splicing of intronic variants that we previously identified by next generation sequencing of the whole CFTR locus. Phenotypic data were collected in 19 patients with CF and CFTR-RD, in whom one of the deep intronic variants has been detected.RESULTS:Three deep-intronic variants promoted the inclusion of pseudo-exons (PE) in the CFTR transcript, hindering the synthesis of a functional protein. The c.2989-313A > T variant, detected in four patients with CF or CFTR-RD from three different families, led to the inclusion of a 118 bp PE. The c.3469-1304C > G variant promoted the inclusion of a 214 bp-PE and was identified in five patients with CF from four families. Haplotype analysis confirmed that this variant was associated with one CF chromosome of African origin. The most represented variant in our cohort was the c.3874-4522A > G, detected in 10 patients with various phenotypes, from male infertility to CF with pancreatic insufficiency.CONCLUSION:These three deep intronic CFTR variants are associated with a large phenotypic spectrum, including typical CF. They should be included in CF diagnostic testing and carrier screening strategies.

Resume Le depistage neonatal (DNN) de la mucoviscidose, grâce a une prise en charge multidisciplinaire tres precoce des nourrissons, est optimal en termes de pronostic pour les patients. Depuis 20 ans, il a connu une expansion internationale spectaculaire. Les performances du DNN national francais realise depuis 2002 sont en accord avec les standards europeens pour la valeur predictive positive (0,31 pour un minimum de 0,30) et la specificite (0,95 pour un minimum de 0,95) ; nous soulignons le nombre tres faible de cas non conclus, un pourcentage tres eleve de test de la sueur realises (95,5 %) et d’identification des mutations (96,6 %), un ratio de cas de mucoviscidose par rapport aux cas de diagnostic incertain de 6,3:1, ainsi qu’une strategie efficace pour reperer les faux negatifs. Une nouvelle organisation du DNN francais vient de se mettre en place, il est capital de maintenir l’efficacite du processus, du nouveau-ne en maternite jusqu’au diagnostic dans des centres de reference ou de competences de la mucoviscidose avec un recueil exhaustif des donnees et leur validation. Par ailleurs, une proposition de changement d’algorithme introduisant le dosage du polypeptide d’activation pancreatique est soumise aux nouvelles instances du DNN. L’annonce d’un diagnostic positif reste difficile et peu standardisee, si bien qu’une plateforme de simulation est en cours de mise en place. Nous detaillons ici les bonnes pratiques et les difficultes de realisation du test de la sueur, avec les seuils de chlorures recemment redefinis, ainsi que les modalites du conseil genetique.

International audience; Background: Minigenes and in silico prediction tools are commonly used to assess the impact on splicing of CFTR variants. Exon skipping is often neglected though it could impact the efficacy of targeted therapies. The aim of the study was to identify exon skipping associated with CFTR variants and to evaluate in silico predictions of seven freely available software.Methods: CFTR basal exon skipping was evaluated on endogenous mRNA extracted from non-CF nasal cells and on two CFTR minigene banks. In silico tools and minigene systems were used to evaluate the impact of CFTR exonic variants on exon skipping.Results: Data showed that out of 65 CFTR variants tested, 26 enhanced exon skipping and that in silico prediction efficacy was of 50%-66%. Some in silico tools presented predictions with a bias towards the occurrence of splicing events while others presented a bias towards the absence of splicing events (non-detection including true negatives and false negatives). Classification of exons depending on their basal exon skipping level increased prediction rates up to 80%.Conclusion: This study indicates that taking basal exon skipping into account could orientate the choice of the in silico tools to improve prediction rates. It also highlights the need to validate effects using in vitro assays or mRNA studies in patients. Eventually, it shows that variant-guided therapy should also target exon skipping associated with variants.

BACKGROUND: The clinical spectrum associated with cystic fibrosis transmembrane conductance regulator (CFTR) variant p.Arg117His is highly variable, ranging from full-blown cystic fibrosis (CF) in a small number of cases to CFTR-related disorders (CFTR-RDs) or no symptoms at all. Therefore, taking into account phenotype variability is essential for interpretation. External quality assessment (EQA) schemes can help laboratories to objectively assess the quality of genotyping and reporting by the laboratory. METHODS: We performed a retrospective longitudinal data analysis on laboratory performance regarding the interpretation of p.Arg117His during CF EQA scheme participation. Completeness and accuracy of reporting on two mock clinical cases were each compared over time (case 1: 2005, 2007 and 2012; case 2: 2015 and 2018). These cases concerned subjects compound heterozygous for p.Phe508del and p.Arg117His in cis with 7T, but with different clinical backgrounds (family planning (case 1) versus diagnostic testing for a child (case 2)). Furthermore, we analyzed the influence of previous participations, annual test volume, accreditation status and laboratory setting on overall performance. RESULTS: Overall performance improved over time, except during the 2007 CF EQA scheme. In addition, previous participations had a beneficial effect on laboratory performance. Accreditation status, annual test volume and laboratory setting did not significantly influence total interpretation scores. CONCLUSIONS: In general, laboratories performed well on both cases, although reporting on the variable clinical spectrum of p.Arg117His in cis with 7T and on the disease liability of individual CFTR variants can still improve. Moreover, this study underlined the educational role of CF EQA schemes. ispartof: JOURNAL OF CYSTIC FIBROSIS vol:19 issue:6 pages:969-974 ispartof: location:Netherlands status: published

International audience; Background: Major issues of newborn screening (NBS) for CF are the assessment of disease liability of variants and of the penetrance of clinical CF, notably in inconclusive diagnosis. The penetrance of CF is defined as the risk of a particular genotype to lead to a CF phenotype.Methods: We aimed to get insight into the penetrance of CF for fifteen CFTR variants: 5 frequent CF-causing and 10 classified as of varying clinical consequence (VCC) or associated with a CFTR-related disorder (CFTR-RD) in CFTR2 or CFTR-France databases. The penetrance was approached by: (1) comparison of variant allelic frequencies in CF patients (CFTR2) and in the general population; (2) estimation of the likelihood of a positive NBS test for the 14 compound heterozygous with F508del and the F508del homozygous genotypes, defined as the ratio of detected/expected number of neonates with a given genotype in the 2002-2017 period.Results: A full penetrance was observed for severe CF-causing variants. Five variants were more frequently found in the general population than in CF patients: TG11T5, TG12T5, TG13T5, L997F and R117H;T7. The likelihood of a positive NBS test was 0.03% for TG11T5, 0.3% for TG12T5, 1.9% for TG13T5, 0.6% for L997F, 11.7% for D1152H, and 17.8% for R117H;T7. Penetrance varied greatly for variants with discrepant classification between CFTR2 and CFTR-France: 5.1% for R117C, 12.3% for T338I, 43.5% for D110H and 52.6% for L206W.Conclusion: These results illustrate the contribution of genetics population data to assess the disease liability of variants for diagnosis and genetic counselling purposes.

International audience; BACKGROUND:Congenital absence of vas deferens (CAVD) represents a major cause of obstructive azoospermia and is mainly related to biallelic alteration of the CFTR gene, also involved in cystic fibrosis. Using whole exome sequencing, we recently identified hemizygous loss-of-function mutations in the Adhesion G Protein-coupled Receptor G2 gene (ADGRG2) as responsible of isolated CAVD in the absence of associated unilateral renal agenesis.OBJECTIVES:The objective of this study was to retrospectively perform ADGRG2 sequencing on a large cohort of patients with CAVD, and 0 or only 1 CFTR defective allele identified after comprehensive testing in order to (a) define more precisely the spectrum and the frequency of ADGRG2 mutations within Caucasian population (b) explore the possibility of co-occurrence of CFTR and ADGRG2 mutations.MATERIALS AND METHODS:We collected 53 DNA samples from CAVD patients with 0 (n = 23) or 1 (n = 30) alteration identified after comprehensive CFTR testing in order to perform ADGRG2 sequencing. Twenty patients had normal ultrasonographic renal examination, and renal status was not documented for 33 patients.RESULTS:We identified six new truncating ADGRG2 mutations in 8 patients including two twin brothers: c.251C > G (p.Ser84*), c.1013delC (p.Pro338Hisfs*4), c.1460delG (p.Gly487Alafs*9), c.2096dupT (p.Phe700Ilefs*29), c.2473C > T (p.Arg825*), and c.1731_1839 + 373del (p.Asn578Thrfs*12), which is a 596 base pair deletion affecting the last five bases of exon 21 and the whole exon 22. Five of the eight patients also harbored an heterozygous CFTR mutation which we consider as incidental regarding the high penetrance expected for ADGRG2 truncating variants. The frequency of ADGRG2 truncating mutation was 26% (5/19 unrelated patients) when presence of both kidneys was attested by ultrasonography and 6.1% (2/33) among patients with unknown renal status.DISCUSSION & CONCLUSION:Our results confirm the interest of ADGRG2 sequencing in patients with CAVD not formerly related to CFTR dysfunction, especially in the absence of associated unilateral renal agenesis.

International audience; Exome sequencing used for molecular diagnosis of Mendelian disorders considerably increases the number of missense variants of unclear significance, whose pathogenicity can be assessed by a variety of prediction tools. As the performance of algorithms may vary according to the datasets, complementary specific resources are needed to improve variant interpretation. As a model, we were interested in the cystic fibrosis transmembrane conductance regulator gene (CFTR) causing cystic fibrosis, in which at least 40% of missense variants are reported. Cystic fibrosis missense analysis (CYSMA) is a new web server designed for online estimation of the pathological relevance of CFTR missense variants. CYSMA generates a set of computationally derived data, ranging from evolutionary conservation to functional observations from three-dimensional structures, provides all available allelic frequencies, clinical observations, and references for functional studies. Compared to software classically used in analysis pipelines on a dataset of 141 well-characterized missense variants, CYSMA was the most efficient tool to discriminate benign missense variants, with a specificity of 85%, and very good sensitivity of 89%. These results suggest that such integrative tools could be adapted to numbers of genes involved in Mendelian disorders to improve the interpretation of missense variants identified in the context of diagnosis.

International audience

Background: Cystic fibrosis (CF) can be revealed during fetal life by diverse ultrasound digestive abnormalities (USDA) such as fetal echogenic bowel or fetal intestinal loop dilatation, nonvisualization of the fetal gallbladder (NVFGB) being rarely observed in isolation. Only 6 cases of CF revealed by isolated NVFGB have been reported so far in the literature. Furthermore, recent studies suggested that this sign is of poor predictive value for CF. Methods: We report on the results of a 6-year French tricenter study on 1,124 cases of fetal USDA for whom a comprehensive molecular study was performed for CF. Results: Among the 37 CF fetuses, 5 (13.5%) presented with isolated NVFGB at ultrasound (US) examination at 24–31 weeks of gestation. This sign was more frequently observed in CF fetuses than in non-CF fetuses, with a likelihood ratio of 2.7. The genotypes included three c.1521_1523del (F508del) homozygous cases and two compound heterozygous cases for a frequent and a rare CF-causing variant. Discussion: These observations highlight the importance to report on the presence and aspect of the fetal gallbladder at the second trimester US scan and to consider prenatal CFTR molecular analysis in cases of isolated NVFGB.

International audience; Whole-exome/genome sequencing analyses lead to detect disease-causing variants that are unrelated to the initial clinical question. Irrespective of any actionable gene list, only pathogenic variants should be considered. The pathogenicity of 55 cystic fibrosis transmembrane conductance regulator (CFTR) variants of known various impacts was assessed by a group of experts by comparing data from specialized databases CFTR-France and CFTR2 with those of general clinical databases ClinVar and Human Gene Mutation Database (HGMD®) Professional and data aggregators VarSome and InterVar. The assessment of cystic fibrosis (CF) variants was correct with ClinVar and HGMD® Professional while less reliable with VarSome and InterVar. Conversely, the risk of overclassifying variants as CF-causing was up to 82% with HGMD® Professional. The concordance between data aggregators was only 50%. The use of general databases and aggregators is thus associated with a substantial risk of misclassifying variants. This evaluation may be extrapolated to other disease conditions and incites to remain cautious in interpreting and disclosing incidental findings.

Background: The CFTR gene is responsible for the autosomal recessive disease Cystic Fibrosis (CF) and CFTR-related disorders (CFTR-RD). More than 2000 variants have been reported. However, the CFTR genotype remains incomplete (with one or none disease-causing variants) in 1% of CF typical cases and even more in CFTR-RD. Aims and Objectives: To find the second disease causing-variant in unsolved cases of CF and CFTR-RD with new methods and assess their biological impact. Methods: We looked for deep intronic variants by studying the whole CFTR locus by Next Generation Sequencing. Impact of variants on splicing was confirmed by in vitro minigene experiments and analysis of CFTR transcripts from patients’ nasal cells. Imaging, spirometric, and biological data (sweat test values, trypsinemia in neonates) were collected. Results: We identified two novel deep intronic CFTR variants that create new splice sites leading to pseudo-exon inclusion and the apparition of a premature stop codon, in 14 patients from 11 families. Ten had bronchiectasis, of whom four had pulmonary infection with atypical germs. Two patients (>40 y/o) had pulmonary surgery. Five patients had normal respiratory function (FEV1% >100) including one with bronchiectasis and pancreatic insufficiency, the two youngest patients ( Conclusion: The two new deep intronic variants are associated with a large phenotypic spectrum, from male infertility to typical CF. These variants should be systematically tested in patients with intermediate or positive sweat tests who do not carry two disease-causing CFTR variants after routine analysis.

International audience; Cystic Fibrosis is among the first diseases to have general population genetic screening tests and one of the most common indications of prenatal and preimplantation genetic diagnosis for single gene disorders. During the past twenty years, thanks to the evolution of diagnostic techniques, our knowledge of CFTR genetics and pathophysiological mechanisms involved in cystic fibrosis has significantly improved. Areas covered: Sanger sequencing and quantitative methods greatly contributed to the identification of more than 2,000 sequence variations reported worldwide in the CFTR gene. We are now entering a new technological age with the generalization of high throughput approaches such as Next Generation Sequencing and Droplet Digital PCR technologies in diagnostics laboratories. These powerful technologies open up new perspectives for scanning the entire CFTR locus, exploring modifier factors that possibly influence the clinical evolution of patients, and for preimplantation and prenatal diagnosis. Expert commentary: Such breakthroughs would, however, require powerful bioinformatics tools and relevant functional tests of variants for analysis and interpretation of the resulting data. Ultimately, an optimal use of all those resources may improve patient care and therapeutic decision-making.

Summary Neonatal screening for cystic fibrosis (CF) can detect infants with elevated immunoreactive trypsinogen (IRT) levels and inconclusive sweat tests and/or CFTR DNA results. These cases of uncertain diagnosis are defined by (1) either the presence of at most one CF-associated cystic fibrosis transmembrane conductance regulator (CFTR) mutation with sweat chloride values between 30 and 59 mmol/L or (2) two CFTR mutations with at least one of unknown pathogenic potential and a sweat chloride concentration below 60 mmol/L. This encompasses various clinical situations whose progression cannot be predicted. In these cases, a sweat chloride test has to be repeated at 12 months, and if possible at 6 and 24 months of life along with extended CFTR sequencing to detect rare mutations. When the diagnosis is not definite, CFTR functional explorations may provide a better understanding of CFTR dysfunction. The initial evaluation of these infants must be conducted in dedicated CF reference centers and should include bacteriological sputum analysis, chest radiology, and fecal elastase assay. The primary care physicians in charge of these patients should be familiar with the current management of CF and should work in collaboration with CF centers. A follow-up should be performed in a CF reference center at 3, 6, and 12 months of life and every year thereafter. Any symptom indicative of CF requires immediate reevaluation of the diagnosis. These guidelines were established by the “neonatal screening and difficult diagnoses” working group of the French CF society. Their objective is to standardize the management of infants with unclear diagnosis.

International audience; Most of the 2,000 variants identified in the CFTR (cystic fibrosis transmembrane regulator) gene are rare or private. Their interpretation is hampered by the lack of available data and resources, making patient care and genetic counseling challenging. We developed a patient-based database dedicated to the annotations of rare CFTR variants in the context of their cis- and trans-allelic combinations. Based on almost 30 years of experience of CFTR testing, CFTR-France (https://cftr.iurc.montp.inserm.fr/cftr) currently compiles 16,819 variant records from 4,615 individuals with cystic fibrosis (CF) or CFTR-RD (related disorders), fetuses with ultrasound bowel anomalies, newborns awaiting clinical diagnosis, and asymptomatic compound heterozygotes. For each of the 736 different variants reported in the database, patient characteristics and genetic information (other variations in cis or in trans) have been thoroughly checked by a dedicated curator. Combining updated clinical, epidemiological, in silico, or in vitro functional data helps to the interpretation of unclassified and the reassessment of misclassified variants. This comprehensive CFTR database is now an invaluable tool for diagnostic laboratories gathering information on rare variants, especially in the context of genetic counseling, prenatal and preimplantation genetic diagnosis. CFTR-France is thus highly complementary to the international database CFTR2 focused so far on the most common CF-causing alleles.

International audience; BACKGROUND:Analysis of cell-free foetal DNA (cff-DNA) in maternal plasma is very promising for early diagnosis of monogenic diseases; in particular, cystic fibrosis (CF). However, NIPD of single-gene disorders has been limited by the availability of suitable technical platforms and the need to set up patient or disease-specific custom-made approaches.METHODS:To make research applications more readily accessible to the clinic, we offer a simple assay combining two independent methods to determine the presence or absence of paternally inherited foetal allele p.Phe508del (the most frequent mutation in CF patients worldwide). The first method detects the presence or absence of a p.Phe508del allele by Mutant Enrichment with 3'-Modified Oligonucleotide PCR coupled to Fragment Length Analysis (MEMO-PCR-FLA). The second method detects the p.Phe508del allele with classical Multiplex Fluorescent PCR including five intragenic and extragenic STR markers of the CFTR locus and a specific SRY sequence.RESULTS:We collected 24 plasma samples from 23 women carrying foetuses at risk for CF and tested each sample using both methods. Our new procedures were successfully applied to 10 couples where fathers carried the p.Phe508del mutation and mothers were carrying a different mutation in the CFTR gene. These simple tests provided clear positive or negative results from the maternal plasma of the pregnant women. We confirmed the presence of cff-DNA in the studied samples by the identification of a tri-allelic DNA profile using a miniSTR kit. All results were correlated with chorionic villus sampling or amniocentesis analyses.CONCLUSIONS:This NIPD approach, easily set up in any clinical laboratory where prenatal diagnosis is routinely performed, offers many advantages over current methods: it is simple, rapid, and cost-effective. It opens up the possibility for testing a large number of couples with offspring at risk for CF.

Motivated by the claimed possibility of a large contribution of the first radial excitation of the $D^{(\ast )}$ to the $B$ semileptonic decay into charmed mesons, also invoked to solve the "$1/2$ vs. $3/2$ semileptonic puzzle", we discuss the transitions to heavy-light radial excitations by a heavy $b \to c$ quark current. We first consider a HQET sum rule, which provides a bound on the slopes of Isgur-Wise functions which we then calculate in the Bakamjian-Thomas framework which both guaranties covariance in the heavy quark limit and satisfies a set of HQET sum rules. We observe a remarkable property that for a large variety of wave functions the transition to the first radial excitation is very small while the transition matrix element to the second radial excitation is large and dominant in saturating the HQET sum rule. This is opposite to what is found in non-relativistic models, where the transition to the first radial excitation dominates the sum rule. The relative magnitude of the transition to the second excitation appears to be weakly dependent on the dynamical scale (radius of the bound states), and the same holds true for the slope of the elastic transition. These features could be tested in the heavy mass limit of lattice QCD. This pattern is shown to be related to the general structure of the Bakamjian-Thomas model, it is independent of the spin structure of the approach and derives mainly from the Lorentz transformation of the spatial wave function, a feature often disregarded in quark models., Comment: 21 pages

This review is based partly on complete articles and partly on abstracts. Three of the 60 articles deal with the total uptake of elements in strawberry plant organs in two different strawberry production systems, both considered as optimal concerning amount and balance of elements. The effect on fruit quality may be dramatic if the level of a particular element is outside this range, but there may also be effects initiated by differences within the optimal range of elements. Most articles refer to product oriented quality, but some focus on consumer oriented quality, as discussed by Shewfelt (1999). The discussion here is on a general basis, so one should keep in mind that there are cultivar differences and that specification of nutrition ideally should mirror the needs of a single cultivar, or a group of cultivars with similar requirements. Also, to get a complete understanding of the subject future reviews should embrace a broader access of information including the effect on plant development ...

Neonatal screening for cystic fibrosis (CF) may detect infants with elevated immunoreactive trypsinogen (IRT) levels but with inconclusive sweat tests and/or DNA results. This includes cases associating (1) either the presence of at most one CF-causing mutation and sweat chloride values between 30 and 59mmol/L or (2) two CFTR mutations with at least one of unknown pathogenicity and a sweat chloride below 60mmol/L. This encompasses different clinical situations whose progression cannot be predicted. These cases require redoing the sweat test at 12 months and if possible at 6 and 24 months of life. This must be associated with extended genotyping. CFTR functional explorations can also help by investigating CFTR dysfunction. These infants must be initially evaluated in dedicated CF centers including bacteriological sputum analysis, chest radiology and fecal elastase dosage. A home practitioner must be informed of the specificity of follow-up. These infants will be reviewed in the CF center at 3, 6 and 12 months and every year. Any CF-related symptom requires reevaluation of the diagnosis. These guidelines were established by the "neonatal screening and difficult diagnoses" working group of the French CF Society. They aim to standardize management of infants with unclear diagnosis in French CF centers.

The Bakamjian-Thomas relativistic quark model, describing hadrons with a fixed number of constituents, yields in the heavy quark limit of QCD covariant Isgur-Wise functions and satisfies the whole tower of lowest moment sum rules (Bjorken-Uraltsev type sum rules). We first recall, as well as earlier results, the new formalism presented in our recent papers on Lorentz representations, which provide an elegant framework for the analysis of this model in the heavy quark limit and stress the results which have been already obtained in this direction. Then, we give some very explicit demonstrations of the fact that the Bakamjian-Thomas framework satifies the sum rules by considering simple cases of Isgur-Wise functions. In addition to the specific Bjorken and Uraltsev sum rules, an important sum rule that involves only heavy mesons with light cloud $j^P = {1 \over 2}^-$ and their radial excitations is demonstrated. This latter sum rule is phenomenologically interesting because it constrains the derivatives of the radially excited Isgur-Wise functions at zero recoil. On the other hand, we recall the limitations of the Bakamjian-Thomas scheme. At finite mass, current matrix elements with the current coupled to the heavy quark are no longer covariant, and higher moment sum rules that hold in the heavy quark limit of QCD are not satisfied.

International audience; The detection of two frequent CFTR disease-causing variations in the context of a newborn screening program (NBS) usually leads to the diagnosis of cystic fibrosis (CF) and a relevant genetic counseling in the family. In the present study, CF-causing variants p.Phe508del (F508del) and c.3140-26A>G (3272-26A>G) were identified on a neonate with positive ImmunoReactive Trypsinogen test by the Elucigene™ CF30 kit. The CF diagnosis initially suggested, despite three inconclusive Sweat Chloride Tests (SCT), was finally ruled out after the familial segregation study combined with a negative SCT. Haplotype studies, based on the comparison of 80 p.Phe508del haplotypes, suggested a probable de novo occurrence of c.3140-26A>G on the p.Phe508del ancestral allele in this family. This false positive case emphasizes the importance of SCT in the NBS strategy. Moreover, it raises the need for familial segregation studies in CF and in overall molecular diagnosis strategy of autosomal recessive diseases.

The OPE treatment that has been so successful in describing inclusive $\bar B \to l \bar \nu X_c$ decays yields sum rules (in particular the Uraltsev sum rule and its higher moments) implying the dominance of the $P$ wave $j_q = 3/2$ charm states in $X_c$ over their $j_q=1/2$ counterparts. This prediction is supported by other general arguments as well as quark model calculations, which illustrate the OPE results, and by preliminary lattice findings. Its failure would indicate a significant limitation in our theoretical understanding of $\bar B \to l \bar \nu X_c$. Some experimental issues have been clarified since a preliminary version of this note had appeared, yet the verdict on the composition of the final states {\em beyond} $D$, $D^*$ and the two narrow $j_q = 3/2$ resonances remains unsettled. Establishing which hadronic configurations -- $D/D^* + \pi, D/D^* + 2 \pi, ...$ -- contribute, what their quantum numbers are and their mass distributions will require considerable experimental effort. We explain the theoretical issues involved and why a better understanding of them will be of significant value. Having significant contributions from a mass continuum distribution below 2.5 GeV raises serious theoretical questions for which we have no good answer. Two lists are given, one with measurements that need to be done and one with items of theoretical homework. Some of the latter can be done by employing existing theoretical tools, whereas others need new ideas.

International audience; PURPOSE:Although 97-99% of CFTR mutations have been identified, great efforts must be made to detect yet-unidentified mutations.METHODS:We developed a small-scale next-generation sequencing approach for reliably and quickly scanning the entire gene, including noncoding regions, to identify new mutations. We applied this approach to 18 samples from patients suffering from cystic fibrosis (CF) in whom only one mutation had hitherto been identified.RESULTS:Using an in-house bioinformatics pipeline, we could rapidly identify a second disease-causing CFTR mutation for 16 of 18 samples. Of them, c.1680-883A>G was found in three unrelated CF patients. Analysis of minigenes and patients' transcripts showed that this mutation results in aberrantly spliced transcripts because of the inclusion of a pseudoexon. It is located only three base pairs from the c.1680-886A>G mutation (1811+1.6kbA>G), the fourth most frequent mutation in southwestern Europe. We next tested the effect of antisense oligonucleotides targeting splice sites on these two mutations on pseudoexon skipping. Oligonucleotide transfection resulted in the restoration of the full-length, in-frame CFTR transcript, demonstrating the effect of antisense oligonucleotide-induced pseudoexon skipping in CF.CONCLUSION:Our data confirm the importance of analyzing noncoding regions to find unidentified mutations, which is essential to designing targeted therapeutic approaches.

International audience; Molecular diagnosis of cystic fibrosis is based on the detection of mutation in the CFTR gene, identified in 1989. During the past 20 years, thanks to evolutions of diagnostic techniques, our knowledge of mutation spectrum and pathophysiological mechanisms involved in the disease has significantly improved. Sanger sequencing and quantitative methods greatly contributed to the identification of the 2,000 sequence variations reported worldwide in CFTR. We are now entering the new technological age with the generalisation of Next Generation Sequencing (NGS) technologies in diagnostics laboratories. These high throughput approaches allow scanning for the entire CFTR locus, including deep intronic regions, and in parallel other candidate genes that possibly influence the clinical evolution of patients. However, this powerful technology poses new challenge in test interpretation. In this chapter, we review the current and new technologies used in molecular diagnostics of cystic fibrosis, particularly NGS approaches. We also present current and new bioinformatics tools available for the interpretation of variants and in vitro/ex vivo and in vivo techniques that can be used to improve the characterization of the functional impact of CFTR variations.

Analysis of circulating cell-free fetal DNA (cffDNA) in maternal plasma is very promising for early diagnosis of monogenic diseases. However, this approach is not yet available for routine use and remains technically challenging because of the low concentration of cffDNA, which is swamped by the overwhelming maternal DNA.To make clinical applications more readily accessible, we propose a new approach based on mutant enrichment with 3′-modified oligonucleotides (MEMO) PCR along with real-time PCR to selectively amplify from the maternal blood the paternally inherited fetal allele that is not present in the maternal genome.The first proof of concept of this strategy was displayed for cystic fibrosis by the accuracy of our detection of the p.Gly542* mutation used as the initial developmental model. Subsequently, a retrospective study of plasmas originating from two pregnant women carrying a fetus with private mutation confirmed the effectiveness of our method. We confirmed the presence of cffDNA in the studied samples by the identification of a tri-allelic DNA profile using a miniSTR kit.This new non-invasive prenatal diagnosis test offers numerous advantages over current methods: it is simple, cost effective, time efficient and does not require complex equipment or bioinformatics settings. Moreover, our assays for different private mutations demonstrate the viability of this approach in clinical settings for monogenic disorders.

The Bakamjian-Thomas relativistic quark model for hadron current matrix elements, while non-covariant at finite mass, is successful in the heavy quark limit : form factors are covariant and satisfy Isgur-Wise scaling and Bjorken-Uraltsev sum rules. Motivated by the so-called "1/2 vs. 3/2 puzzle" in B decays to positive parity D**, we examine the implications of the model at finite mass. In the elastic case 1/2^- -> 1/2^-, the HQET constraints for the O(1/m_Q) corrections are analytically fulfilled. A number of satisfying regularities is also found for inelastic transitions. We compute the form factors using the wave functions given by the Godfrey-Isgur potential. For 1/2^- \to 3/2^+ the departures from the heavy quark limit are small, but we find a strong enhancement in 1/2^- -> 1/2^+ (for 0^- -> 0^+). This enhancement is linked to a serious difficulty of the model at finite mass for the inelastic transitions, namely a violation of the HQET constraints at zero recoil formulated by Leibovich et al. These are nevertheless satisfied in the non-relativistic limit for the light quark. We conclude that these HQET rigorous constraints are crucial in the construction of a sensible relativistic quark model of inelastic form factors.

National audience; New indicators for bioavailability, in link with the knowledge of the phenomena involved in plants' phosphate nutrition, were assessed in carrot crops, and experimental references were established. The trials conducted in the Aquitaine region, a major French carrot production area for the fresh market, consisted in analysing the response of carrot, in terms of yield, to a wide range of soil phosphorus supply levels. The synthesis of the data acquired during a three-year study shows the importance of the P concentration in the soil solution, i.e. the soluble P, noted as Cp, for diagnosing the soil's nutritional status. Measuring Cp may be sufficient in the case of slightly buffered soils, characteristic for carrot cultivation in Aquitaine. However, the quality of the relation between yield and soil supply improves when it is represented by the sum of soluble P and diffusible P, linked to the plant's functioning. This is indicator E. Threshold values were determined, corresponding to 1 mg P L-1 and 15.7 mg P kg-1 soil respectively for Cp and E. Those thresholds require further validation in a wider range of agronomic conditions. Furthermore, the newly acquired knowledge and technical references already make it possible to rectify fertilising practices in Aquitaine. In optimum fertilising conditions, the crop takes up 26 kg P ha-1, i.e. 60 kg P2O5 ha-1, and soil supply represents two thirds of the phosphorus uptake. This study offers perspective for new diagnosing methods, which take into account the functioning of the plant species. It shows that a more sparing use of phosphorus in carrot crops is possible without compromising yield. Changing fertilising practices accordingly will guarantee a better preservation of natural resources and water quality. Finally, the outcome of this study encourages us to apply those new concepts in soil phosphorus supply to a wider range of vegetables, in order to establish a reliable basis for integrated P management practices.

International audience; The genetic mechanisms that regulate CFTR, the gene responsible for cystic fibrosis, have been widely investigated in cultured cells. However, mechanisms responsible for tissue-specific and time-specific expression are not completely elucidated in vivo. Through the survey of public databases, we found that the promoter of CFTR was associated with bivalent chromatin in human embryonic stem (ES) cells. In this work, we analyzed fetal (at different stages of pregnancy) and adult tissues and showed that, in digestive and lung tissues, which expressed CFTR, H3K4me3 was maintained in the promoter. Histone acetylation was high in the promoter and in two intronic enhancers, especially in fetal tissues. In contrast, in blood cells, which did not express CFTR, the bivalent chromatin was resolved (the promoter was labeled by the silencing mark H3K27me3). Cis-regulatory sequences were associated with lowly acetylated histones. We also provide evidence that the tissue-specific expression of CFTR is not regulated by dynamic changes of DNA methylation in the promoter. Overall, this work shows that a balance between activating and repressive histone modifications in the promoter and intronic enhancers results in the fine regulation of CFTR expression during development, thereby ensuring tissue specificity.

We show that the sum rule recently proved by Uraltsev in the heavy quark limit of QCD holds in relativistic quark models \`a la Bakamjian and Thomas, that were already shown to satisfy Isgur-Wise scaling and Bjorken sum rule. This new sum rule provides a {\it rationale} for the lower bound of the slope of the elastic IW function $\rho^2 \geq {3 \over 4}$ obtained within the BT formalism some years ago. Uraltsev sum rule suggests an inequality $|\tau_{3/2}(1)| > |\tau_{1/2}(1)|$. This difference is interpreted in the BT formalism as due to the Wigner rotation of the light quark spin, independently of a possible LS force. In BT models, the sum rule convergence is very fast, the $n = 0$ state giving the essential contribution in most of the phenomenological potential models. We underline that there is a serious problem, in the heavy quark limit of QCD, between theory and experiment for the decays $B \to D^*_{0,1}(broad) \ell \nu$, independently of any model calculation., Comment: 16 pages, Latex

Glycosylation represents an attractive target for protein engineering of novel antibiotics, because specific attachment of one or more deoxysugars is required for the bioactivity of many antibiotic and antitumour polyketides. However, proper assessment of the potential of these enzymes for such combinatorial biosynthesis requires both more precise information on the enzymology of the pathways and also improved Escherichia coli-actinomycete shuttle vectors. New replicative vectors have been constructed and used to express independently the dnmU gene of Streptomyces peucetius and the eryBVII gene of Saccharopolyspora erythraea in an eryBVII deletion mutant of Sac. erythraea. Production of erythromycin A was obtained in both cases, showing that both proteins serve analogous functions in the biosynthetic pathways to dTDP-L-daunosamine and dTDP-L-mycarose, respectively. Over-expression of both proteins was also obtained in S. lividans, paving the way for protein purification and in vitro monitoring of enzyme activity. In a further set of experiments, the putative desosaminyltransferase of Sac. erythraea, EryCIII, was expressed in the picromycin producer Streptomyces sp. 20032, which also synthesises dTDP-D-desosamine. The substrate 3-alpha-mycarosylerythronolide B used for hybrid biosynthesis was found to be glycosylated to produce erythromycin D only when recombinant EryCIII was present, directly confirming the enzymatic role of EryCIII. This convenient plasmid expression system can be readily adapted to study the directed evolution of recombinant glycosyltransferases.

We explicitly check quark-hadron duality to order $(m_b-m_c)\Lambda/m_b^2$ for $b \to c l\nu$ decays in the limit $m_b-m_c \ll m_b$ including ground state and orbitally excited hadrons. Duality occurs thanks to a new sum rule which expresses the subleading HQET form factor $\xi_3$ or, in other notations, $a_+^{(1)}$ in terms of the infinite mass limit form factors and some level splittings. We also demonstrate the sum rule, which is not restricted to the condition $m_b-m_c \ll m_b$, applying OPE to the longitudinal axial component of the hadronic tensor without neglecting the $1/m_b$ subleading contributions to the form factors. We argue that this method should produce a new class of sum rules, depending on the current, beyond Bjorken, Voloshin and the known tower of higher moments. Applying OPE to the vector currents we find another derivation of the Voloshin sum rule. From independent results on $\xi_3$ we derive a sum rule which involves only the $\tau_{1/2}^{(n)}$ and $\tau_{3/2}^{(n)}$ form factors and the corresponding level splittings. The latter strongly supports a theoretical evidence that the $B$ semileptonic decay into narrow orbitally-excited resonances dominates over the decay into the broad ones, in apparent contradiction with some recent experiments. We discuss this issue., Comment: 9 pages

Poly(A) polymerase I (PAP I) of Escherichia coli is a member of the nucleotidyltransferase (Ntr) superfamily that includes the eukaryotic PAPs and all the known tRNA CCA-adding enzymes. Five highly conserved aspartic acids in the putative catalytic site of PAP I were changed to either alanine or proline, demonstrating their importance for polymerase activity. A glycine that is absolutely conserved in all Ntrs was also changed yielding a novel mutant protein in which ATP was wastefully hydrolysed in a primer-independent reaction. This is the first work to characterize the catalytic site of a eubacterial PAP and, despite the conservation of certain sequences, we predict that the overall architecture of the eukaryotic and eubacterial active sites is likely to be different. Binding sites for RNase E, a component of the RNA degradosome, and RNA were mapped by North-western and Far-western blotting using truncated forms of PAP I. Additional protein-protein interactions were detected between PAP I and CsdA, RhlE and SrmB, suggesting an unexpected connection between PAP I and these E. coli DEAD box RNA helicases. These results show that the functional organization of PAP I is similar to the eukaryotic PAPs with an N-terminal catalytic domain, a C-terminal RNA binding domain and sites for the interaction with other protein factors.

There has been increased interest in bacterial polyadenylation with the recent demonstration that 3′ poly(A) tails are involved in RNA degradation. Poly(A) polymerase I (PAP I) of Escherichia coli is a member of the nucleotidyltransferase (Ntr) family that includes the functionally related tRNA CCA-adding enzymes. Thirty members of the Ntr family were detected in a search of the current database of eubacterial genomic sequences. Gram-negative organisms from the β and γ subdivisions of the purple bacteria have two genes encoding putative Ntr proteins, and it was possible to predict their activities as either PAP or CCA adding by sequence comparisons with the E. coli homologues. Prediction of the functions of proteins encoded by the genes from more distantly related bacteria was not reliable. The Bacillus subtilis papS gene encodes a protein that was predicted to have PAP activity. We have overexpressed and characterized this protein, demonstrating that it is a tRNA nucleotidyltransferase. We suggest that the papS gene should be renamed cca , following the notation for its E. coli counterpart. The available evidence indicates that cca is the only gene encoding an Ntr protein, despite previous suggestions that B. subtilis has a PAP similar to E. coli PAP I. Thus, the activity involved in RNA 3′ polyadenylation in the gram-positive bacteria apparently resides in an enzyme distinct from its counterpart in gram-negative bacteria.